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Molecular and functional characterization of two novel human C-C chemokines as inhibitors of two distinct classes of myeloid progenitors.

Patel VP, Kreider BL, Li Y, Li H, Leung K, Salcedo T, Nardelli B, Pippalla V, Gentz S, Thotakura R, Parmelee D, Gentz R, Garotta G - J. Exp. Med. (1997)

Bottom Line: Both of the cDNAs were cloned into a baculovirus vector and expressed in insect cells.The mature recombinant MPIF-1 protein consists of 99 amino acids and is most homologous to macrophage inflammatory protein (MIP)-1alpha, showing 51% identity.On eosinophils, MPIF-2 produces a transient rise of cytosolic Ca2+ and uses the receptor for eotaxin and MCP-4.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Human Genome Sciences, Inc., Rockville, Maryland 20850, USA.

ABSTRACT
Two novel human beta-chemokines, Ck beta-8 or myeloid progenitor inhibitory factor 1 (MPIF-1), and Ck beta-6 or MPIF-2, were discovered as part of a large scale cDNA sequencing effort. The MPIF-1 and MPIF-2 cDNAs were isolated from aortic endothelium and activated monocyte libraries, respectively. Both of the cDNAs were cloned into a baculovirus vector and expressed in insect cells. The mature recombinant MPIF-1 protein consists of 99 amino acids and is most homologous to macrophage inflammatory protein (MIP)-1alpha, showing 51% identity. It displays chemotactic activity on resting T lymphocytes and monocytes, a minimal but significant activity on neutrophils, and is negative on activated T lymphocytes. MPIF-1 is also a potent suppressor of bone marrow low proliferative potential colony-forming cells, a committed progenitor that gives rise to granulocyte and monocyte lineages. The mature recombinant MPIF-2 has 93 amino acid residues and shows 39 and 42% identity with monocyte chemoattractant protein (MCP)-3 and MIP-1alpha, respectively. It displays chemotactic activity on resting T lymphocytes, a minimal activity on neutrophils, and is negative on monocytes and activated T lymphocytes. On eosinophils, MPIF-2 produces a transient rise of cytosolic Ca2+ and uses the receptor for eotaxin and MCP-4. In hematopoietic assays, MPIF-2 strongly suppressed the colony formation by the high proliferative potential colony-forming cell (HPP-CFC), which represents a multipotential hematopoietic progenitor.

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Analysis of MPIF-1 (A) and MPIF-2 (B) proteins. SDSPAGE was performed on 5 μg of purified protein under reducing conditions. The gel was stained with Coomassie blue. Lane 1 of each panel  shows the molecular weight standards. C shows the NH2-terminal sequences of MPIF-1 and MPIF-2 proteins as determined by Edman degradation.
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Figure 4: Analysis of MPIF-1 (A) and MPIF-2 (B) proteins. SDSPAGE was performed on 5 μg of purified protein under reducing conditions. The gel was stained with Coomassie blue. Lane 1 of each panel shows the molecular weight standards. C shows the NH2-terminal sequences of MPIF-1 and MPIF-2 proteins as determined by Edman degradation.

Mentions: Recombinant MPIF-1 and MPIF-2 proteins were expressed in Sf9 insect cells infected with a recombinant baculovirus. SDS-PAGE analysis of a purified MPIF-1 preparation revealed a single Coomassie blue–stained band with a molecular mass of 11–12 kD (Fig. 4 A). Edman degradation analysis showed that the MPIF-1 protein was processed to yield a protein with RVTKDAE as the NH2-terminus and an expected molecular mass of ∼11.2 kD, thus confirming that the observed sequence is identical to that predicted from the cDNA sequence (Fig. 4 C). Consistent with the lack of N-glycosylation sites no mannose or N-acetylglucosamine were detected in the MPIF-1 protein.


Molecular and functional characterization of two novel human C-C chemokines as inhibitors of two distinct classes of myeloid progenitors.

Patel VP, Kreider BL, Li Y, Li H, Leung K, Salcedo T, Nardelli B, Pippalla V, Gentz S, Thotakura R, Parmelee D, Gentz R, Garotta G - J. Exp. Med. (1997)

Analysis of MPIF-1 (A) and MPIF-2 (B) proteins. SDSPAGE was performed on 5 μg of purified protein under reducing conditions. The gel was stained with Coomassie blue. Lane 1 of each panel  shows the molecular weight standards. C shows the NH2-terminal sequences of MPIF-1 and MPIF-2 proteins as determined by Edman degradation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196270&req=5

Figure 4: Analysis of MPIF-1 (A) and MPIF-2 (B) proteins. SDSPAGE was performed on 5 μg of purified protein under reducing conditions. The gel was stained with Coomassie blue. Lane 1 of each panel shows the molecular weight standards. C shows the NH2-terminal sequences of MPIF-1 and MPIF-2 proteins as determined by Edman degradation.
Mentions: Recombinant MPIF-1 and MPIF-2 proteins were expressed in Sf9 insect cells infected with a recombinant baculovirus. SDS-PAGE analysis of a purified MPIF-1 preparation revealed a single Coomassie blue–stained band with a molecular mass of 11–12 kD (Fig. 4 A). Edman degradation analysis showed that the MPIF-1 protein was processed to yield a protein with RVTKDAE as the NH2-terminus and an expected molecular mass of ∼11.2 kD, thus confirming that the observed sequence is identical to that predicted from the cDNA sequence (Fig. 4 C). Consistent with the lack of N-glycosylation sites no mannose or N-acetylglucosamine were detected in the MPIF-1 protein.

Bottom Line: Both of the cDNAs were cloned into a baculovirus vector and expressed in insect cells.The mature recombinant MPIF-1 protein consists of 99 amino acids and is most homologous to macrophage inflammatory protein (MIP)-1alpha, showing 51% identity.On eosinophils, MPIF-2 produces a transient rise of cytosolic Ca2+ and uses the receptor for eotaxin and MCP-4.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Human Genome Sciences, Inc., Rockville, Maryland 20850, USA.

ABSTRACT
Two novel human beta-chemokines, Ck beta-8 or myeloid progenitor inhibitory factor 1 (MPIF-1), and Ck beta-6 or MPIF-2, were discovered as part of a large scale cDNA sequencing effort. The MPIF-1 and MPIF-2 cDNAs were isolated from aortic endothelium and activated monocyte libraries, respectively. Both of the cDNAs were cloned into a baculovirus vector and expressed in insect cells. The mature recombinant MPIF-1 protein consists of 99 amino acids and is most homologous to macrophage inflammatory protein (MIP)-1alpha, showing 51% identity. It displays chemotactic activity on resting T lymphocytes and monocytes, a minimal but significant activity on neutrophils, and is negative on activated T lymphocytes. MPIF-1 is also a potent suppressor of bone marrow low proliferative potential colony-forming cells, a committed progenitor that gives rise to granulocyte and monocyte lineages. The mature recombinant MPIF-2 has 93 amino acid residues and shows 39 and 42% identity with monocyte chemoattractant protein (MCP)-3 and MIP-1alpha, respectively. It displays chemotactic activity on resting T lymphocytes, a minimal activity on neutrophils, and is negative on monocytes and activated T lymphocytes. On eosinophils, MPIF-2 produces a transient rise of cytosolic Ca2+ and uses the receptor for eotaxin and MCP-4. In hematopoietic assays, MPIF-2 strongly suppressed the colony formation by the high proliferative potential colony-forming cell (HPP-CFC), which represents a multipotential hematopoietic progenitor.

Show MeSH
Related in: MedlinePlus